Combined Phylogeographic Analyses and Epidemiologic Contact Tracing to Characterize Atypically Pathogenic Avian Influenza (H3N1) Epidemic, Belgium, 2019.

The high economic impact and zoonotic potential of avian influenza call for detailed investigations of dispersal dynamics of epidemics. We integrated phylogeographic and epidemiologic analyses to investigate the dynamics of a low pathogenicity avian influenza (H3N1) epidemic that occurred in Belgium during 2019. Virus genomes from 104 clinical samples originating from 85% of affected farms were sequenced. A spatially explicit phylogeographic analysis confirmed a dominating northeast to southwest dispersal direction and a long-distance dispersal event linked to direct live animal transportation between farms. Spatiotemporal clustering, transport, and social contacts strongly correlated with the phylogeographic pattern of the epidemic. We detected only a limited association between wind direction and direction of viral lineage dispersal. Our results highlight the multifactorial nature of avian influenza epidemics and illustrate the use of genomic analyses of virus dispersal to complement epidemiologic and environmental data, improve knowledge of avian influenza epidemiologic dynamics, and enhance control strategies.

Metagenomic sequencing determines complete infectious bronchitis virus (avian Gammacoronavirus) vaccine strain genomes and associated viromes in chicken clinical samples.

Infectious bronchitis virus (IBV, genus Gammacoronavirus) causes an economically important and highly contagious disease in chicken. Random primed RNA sequencing was applied to two IBV positive clinical samples and one in ovo-passaged virus. The virome of a cloacal swab pool was dominated by IBV (82% of viral reads) allowing de novo assembly of a GI-13 lineage complete genome with 99.95% nucleotide identity to vaccine strain 793B. In addition, substantial read counts (16% of viral reads) allowed the assembly of a near-complete chicken astrovirus genome, while lower read counts identified the presence of chicken calicivirus and avian leucosis virus. Viral reads in a respiratory/intestinal tissue pool were distributed between IBV (22.53%), Sicinivirus (Picornaviridae, 24%), and avian leucosis virus (37.04%). A complete IBV genome with 99.95% nucleotide identity to vaccine strain H120 (lineage GI-1), as well as a near-complete avian leucosis virus genome and a partial Sicinivirus genome were assembled from the tissue sample data. Lower read counts identified chicken calicivirus, Avibirnavirus (infectious bursal disease virus, assembling to 98.85% of segment A and 69.66% of segment B closely related to D3976/1 from Germany, 2017) and avian orthoreovirus, while three avian orthoavulavirus 1 reads confirmed prior real-time RT-PCR result. IBV sequence variation analysis identified both fixed and minor frequency variations in the tissue sample compared to its in ovo-passaged virus. Metagenomic methods allow the determination of complete coronavirus genomes from clinical chicken samples while providing additional insights in RNA virus sequence diversity and coinfecting viruses potentially contributing to pathogenicity.

Atypical Pathogenicity of Avian Influenza (H3N1) Virus Involved in Outbreak, Belgium, 2019.

In 2019, an outbreak of avian influenza (H3N1) virus infection occurred among commercial poultry in Belgium. Full-genome phylogenetic analysis indicated a wild bird origin rather than recent circulation among poultry. Although classified as a nonnotifiable avian influenza virus, it was associated with reproductive tropism and substantial mortality in the field.

Phylogeographic Analysis of African Swine Fever Virus, Western Europe, 2018.

In September 2018, African swine fever in wild boars was detected in Belgium. We used African swine fever-infected spleen samples to perform a phylogenetic analysis of the virus. The causative strain belongs to genotype II, and its closest relatives are viruses previously isolated in Ukraine, Belarus, Estonia, and European Russia.

Domestic canaries (Serinus canaria forma domestica) are susceptible to low pathogenic avian influenza virus infections.

Avian influenza viruses have been isolated from many bird species; however, little is known about the susceptibility of pet birds to low pathogenic avian influenza (LPAI) viruses. To address this research gap, domestic canaries (Serinus canaria forma domestica) were experimentally infected with H5 and H7 LPAI viruses to determine susceptibility and to evaluate samples for diagnostic purposes. Clinical evidence of infection (e.g. ruffled plumage and apathy) and mortality were noted for the canaries inoculated with chicken-adapted LPAI viruses. Real-time reverse transcription-polymerase chain reaction (RRT-PCR) demonstrated higher viral RNA levels in buccal compared to faecal samples. No clinical signs or mortality were observed in canaries inoculated with LPAI virus originating from wild birds; however, the canaries in this group did have evidence of viral RNA in buccal and faecal samples. Overall, this study showed that domestic canaries are susceptible to LPAI virus infections and that they can shed large amounts of viral RNA, primarily through the respiratory route. Thus, buccal swabs might be better samples than faeces for efficient detection of some LPAI virus infections in these birds. Although canaries have not been identified as a significant reservoir for LPAI viruses, they may be infected by LPAI viruses. Thus, the importance of the control of domestic canaries for detection of LPAI viruses should not be underestimated, especially in the contexts of international commercial exchange and outbreaks. RESEARCH HIGHLIGHTS Canaries are susceptible to infection with H5/H7 LPAI viruses. Canaries inoculated with LPAI viruses excrete large amounts of viral RNA. Buccal swabs may be appropriate specimens for AI virus detection in canaries. The control of canaries for LPAI virus detection should not be overlooked.

Characterization of two recombinant HVT-IBD vaccines by VP2 insert detection and cell-mediated immunity after vaccination of specific pathogen-free chickens.

Infectious bursal disease (IBD) is an avian viral disease that causes severe economic losses in the poultry industry worldwide. The live IBD virus (IBDV) has a potential immunosuppressive effect. Currently available IBDV vaccines have shortcomings, prompting the development of safer and more effective vaccination approaches, including the use of the recombinant turkey herpesvirus vaccine expressing the immunogenic structural VP2 protein of IBDV (recombinant HVT (rHVT)-IBD). The objectives of this study were twofold: (i) to develop in vitro assays and molecular tools to detect the VP2 protein and gene and (ii) to evaluate cell-mediated immunity (CMI) induced by rHVT-IBD vaccination of day-old specific pathogen-free chickens. The VP2 protein expressed by rHVT-IBD-infected chicken embryo fibroblasts was detected using the enzyme-linked immunosorbent assay and immunofluorescence. Using molecular techniques, the VP2 gene was detected in various organs, providing a method to monitor vaccine uptake. rHVT-IBD vaccination induced CMI responses in specific pathogen-free chickens at 5 weeks. CMI was detected by measuring chicken interferon-gamma after ex vivo antigenic stimulation of splenocytes. Moreover, our results showed that the enzyme-linked immunospot approach is more sensitive in detecting chicken interferon-gamma than enzyme-linked immunosorbent assay. The tools developed in this study may be useful in the characterization of new-generation recombinant vaccines and the cellular immune response they induce.

Evaluation of the pathogenicity of West Nile virus (WNV) lineage 2 strains in a SPF chicken model of infection: NS3-249Pro mutation is neither sufficient nor necessary for conferring virulence.

Lineage 2 West Nile virus (WNV) strains were reported for the first time in Europe in 2004. Despite an almost silent circulation around their entry point in Hungary, an upsurge of pathogenicity occurred in 2010 as 262 people suffered from neuroinvasive disease in Greece. This increase in virulence was imputed to the emergence of a His249Pro mutation in the viral NS3 helicase, as previously evidenced in American crows experimentally infected with the prototype lineage 1 North-American WNV strain. However, since 2003, WNV strains bearing the NS3Pro genotype are regularly isolated in Western-Mediterranean countries without being correlated to any virulent outbreak in vertebrates. We thus sought to evaluate the weight of the NS3249Pro genotype as a virulence marker of WNV in an in vivo avian model of WNV infection. We therefore characterized three genetically-related Eastern-Europe lineage 2 WNV strains in day-old specific pathogen-free (SPF) chickens: Hun2004 and Aus2008 which are both characterized by a NS3249His genotype, and Gr2011 which is characterized by a NS3249Pro genotype. Unlike Hun2004 and Aus2008, Gr2011 was weakly virulent in SPF chicks as Gr2011-induced viremia was lower and waned quicklier than in the Hun2004 and Aus2008 groups. Overall, this study showed that the presence of a proline residue at position 249 of the viral NS3 helicase is neither sufficient nor necessary to confer pathogenicity to any given lineage 2 WNV strain in birds.

Multiyear Serological Surveillance of Notifiable Influenza A Viruses in Belgian Poultry: A Retrospective Analysis.

Surveillance of notifiable avian influenza (NAI) virus is mandatory in European member states, and each year a serological survey is performed to detect H5 and H7 circulation in poultry holdings. In Belgium, this serological monitoring is a combination of a stratified and a risk-based approach and is applied to commercial holdings with more than 200 birds. Moreover, a competitive nucleoprotein (NP) ELISA has been used as first screening method since 2010. A retrospective analysis of the serological monitoring performed from 2007 through 2013 showed sporadic circulation of notifiable low-pathogenicity avian influenza (LPAI) viruses in Belgian holdings with a fluctuating apparent flock seroprevalence according to years and species. Overall, the highest apparent flock seroprevalence was detected for the H5 subtype in domestic Anatidae, with 20%-50% for breeding geese and 4%-9% for fattening ducks. Positive serology against non-H5/H7 viruses was also observed in the same species with the use of the IDScreen influenza A antibody competition ELISA kit (ID-vet NP ELISA), and confirmed by isolation of H2, H3, H6, and H9 LPAI viruses. Among Galliformes, the apparent flock seroprevalence was lower, ranging between 0.3% and 1.3%. Circulation of notifiable LPAI viruses was only observed in laying hens with a similar seroprevalence for H5 and H7. Based on ID-vet NP ELISA results, no circulation of LPAI viruses, regardless the subtype, was observed in breeding chickens and fattening turkeys. Retrospectively, the use of an ELISA as first-line test not only reduced the number of hemagglutination inhibition tests to be performed, but also gave a broader evaluation of the prevalence of LPAI viruses in general, and might help to identify the most at-risk farms.

Differential Viral Fitness Between H1N1 and H3N8 Avian Influenza Viruses Isolated from Mallards (Anas platyrhynchos).

Homosubtypic and heterosubtypic immunity in mallards (Anas platyrhynchos) play an important role in the avian influenza virus (AIV) diversity. The mechanisms of AIV replication among wild birds and the role of immunity in AIV diversity have thus not been completely clarified. During the monitoring of AI circulation among wild waterfowl in 2007-2008, two viruses (H3N8 and H1N1) were isolated from ducks caught in a funnel trap located in La Hulpe wetland in Belgium. H3N8 viruses were revealed to be more prevalent in the mallard population than was H1N1, which might suggest a better adaptation to this species. In order to investigate this hypothesis, we characterized both isolated viruses biologically by experimental inoculation. Virus excretion and humoral response induced by both isolated viruses were evaluated in mallards after a first infection followed by a homo- or heterosubtypic reinfection under controlled experimental conditions. The H1N1 virus had a delayed peak of excretion of 4 days compared to the H3N8, but the virus shedding was more limited, earlier, and shorter after each reinfection. Moreover, the H3N8 virus could spread to all ducks after homo- or heterosubtypic reinfections and during a longer period. Although the humoral response induced by both viruses after infection and reinfection could be detected efficiently by competitive ELISA, only a minimal H1 antibody response and almost no H3-specific antibodies could be detected by the HI test. Our results suggest that the H3N8 isolate replicates better in mallards under experimental controlled conditions.

A model for the transfer of passive immunity against Newcastle disease and avian influenza in specific pathogen free chickens.

Chicks possess maternally derived antibody (MDA) against pathogens and vaccines previously encountered by the dams. This passive immunity is important in early life, when the immune system is immature and unable to fight off infection. On the other hand, MDA can also affect the development of the immune system and interfere with vaccination against avian diseases such as Newcastle disease (ND) and avian influenza (AI). The effect of MDA is generally investigated by studying the progeny of vaccinated dams, which is time-consuming, poorly flexible and expensive. Moreover, the antibody titres obtained are not homogeneous. In this study, a model was developed to offer a faster, more reproducible and cheaper way to study passive immunity in specific pathogen free chickens by injection of a polyclonal serum into the egg yolk at embryonic day 14, combined with an intraperitoneal injection at day 1. A satisfactory model, with consistent, homogeneous antibody titres, as well as persistence close to natural passive immunity, could be obtained for ND virus. On the other hand, the application of this optimized protocol in an H5 AI context induced only a low artificial passive immunity compared with that described in the literature for the progeny of AI vaccinated dams. This artificial model should facilitate future studies regarding the effect of passive immunity on vaccine efficacy at a young age and its effect on immune system development.

Comparison of single 1-day-old chick vaccination using a Newcastle disease virus vector with a prime/boost vaccination scheme against a highly pathogenic avian influenza H5N1 challenge.

Avian influenza (AI) vaccines should be used as part of a whole comprehensive AI control programme. Vectored vaccines based on Newcastle disease virus (NDV) are very promising, but are so far licensed in only a few countries. In the present study, the immunogenicity and protection against a highly pathogenic H5N1 influenza challenge were evaluated after vaccination with an enterotropic NDV vector expressing an H5 haemagglutinin (rNDV-H5) in 1-day-old specific pathogen free chickens inoculated once, twice or once followed by a heterologous boost with an inactivated H5N9 vaccine (iH5N9). The heterologous prime/boost rNDV-H5/iH5N9 combination afforded the best level of protection against the H5N1 challenge performed at 6 weeks of age. Two rNDV-H5 administrations conferred a good level of protection after challenge, although only a cellular H5-specific response could be detected. Interestingly, a single administration of rNDV-H5 gave the same level of protection as the double administration but without any detectable H5-specific immune response. In contrast to AI immunity, a high humoral, mucosal and cellular NDV-specific immunity could be detected up to 6 weeks post vaccination after using the three different vaccination schedules. NDV-specific mucosal and cellular immune responses were slightly higher after double rNDV-H5 vaccination when compared with single inoculation. Finally, the heterologous prime/boost rNDV-H5/iH5N9 combination induced a broader detectable immunity including systemic, mucosal and cellular AI and NDV-specific responses.

Wildebeest-Derived Malignant Catarrhal Fever: A Bovine Peripheral T Cell Lymphoma Caused by Cross-Species Transmission of Alcelaphine Gammaherpesvirus 1.

Gammaherpesviruses (γHVs) include viruses that can induce lymphoproliferative diseases and tumors. These viruses can persist in the long term in the absence of any pathological manifestation in their natural host. Alcelaphine gammaherpesvirus 1 (AlHV-1) belongs to the genus Macavirus and asymptomatically infects its natural host, the wildebeest (Connochaetes spp.). However, when transmitted to several susceptible species belonging to the order Artiodactyla, AlHV-1 is responsible for the induction of a lethal lymphoproliferative disease, named wildebeest-derived malignant catarrhal fever (WD-MCF). Understanding the pathogenic mechanisms responsible for the induction of WD-MCF is important to better control the risks of transmission and disease development in susceptible species. The aim of this review is to synthesize the current knowledge on WD-MCF with a particular focus on the mechanisms by which AlHV-1 induces the disease. We discuss the potential mechanisms of pathogenesis from viral entry into the host to the maintenance of viral genomes in infected CD8+ T lymphocytes, and we present current hypotheses to explain how AlHV-1 infection induces a peripheral T cell lymphoma-like disease.

Phylogeographic analysis of avian influenza viruses isolated from Charadriiformes in Belgium confirms intercontinental reassortment in gulls.

Nine influenza viruses isolated from gulls and shorebirds in Belgium (2008-2010), including H3N8, H5N2, H6N1, H11N9, H13N6, H13N8, and H16N3 subtypes, were targeted using random amplification and next-generation sequencing. The gene segments of these viruses segregated into three phylogeographic lineage types: (1) segments circulating in waterfowl in Eurasia with sporadic introduction in other species and in the Americas ("Eurasian avian"), (2) segments circulating in American waterfowl with sporadic introduction to other species and regions ("American avian"), and (3) segments circulating exclusively in gulls and shorebirds and having increased connectivity between the two hemispheres ("Charadriiformes specific"). Notably, an H6N1 and an H5N2 isolated from L. argentatus had mainly Eurasian avian genes but shared a matrix segment of American avian origin (first documentation in European gulls of transhemispheric reassortment). These data support the growing evidence of an important role of Charadriiformes birds in the dynamic nature of avian influenza ecology.

Further evidence for the widespread co-circulation of lineages 4b and 7 velogenic Newcastle disease viruses in rural Nigeria.

Newcastle disease (ND) is an endemic disease in rural poultry of Western Africa. It may cause severe economic losses in the poultry sector and, as such, is listed as a notifiable disease by the World Organisation for Animal Health (OIE). Recently, a new genetic lineage of ND viruses was discovered in Western Africa. We determined the complete fusion (F) gene coding sequence of 12 ND viruses isolated from pigeons and rural chickens in six Nigerian states in 2007 and 2008. Phylogenetic analysis of the complete F coding sequence confirmed the circulation of genetically diverse ND isolates in a large geographic area in Nigeria. Next to isolates belonging to lineage 4b, viruses of the recently discovered lineage 7 (some of which were previously reported to escape routine real-time reverse transcriptase-polymerase chain reaction detection) were isolated in six states during the two-year period. The documented genetic variants occurred over a large geographic area, indicating an endemic circulation of these viruses. Three different velogenic fusion gene cleavage site motifs were observed. These findings confirm the endemic circulation and diversification of ND isolates in rural poultry and pigeons in Nigeria and highlight the importance of surveillance in developing countries to monitor the validity of rapid molecular diagnostic tools and of vaccination regimes.

Intranasal DNA vaccination induces potent mucosal and systemic immune responses and cross-protective immunity against influenza viruses.

The induction of potent virus-specific immune responses at mucosal surfaces where virus transmission occurs is a major challenge for vaccination strategies. In the case of influenza vaccination, this has been achieved only by intranasal delivery of live-attenuated vaccines that otherwise pose safety problems. Here, we demonstrate that potent mucosal and systemic immune responses, both cellular and humoral, are induced by intranasal immunization using formulated DNA. We show that formulation with the DNA carrier polyethylenimine (PEI) improved by a 1,000-fold the efficiency of gene transfer in the respiratory track following intranasal administration of luciferase-coding DNA. Using PEI formulation, intranasal vaccination with DNA-encoding hemagglutinin (HA) from influenza A H5N1 or (H1N1)2009 viruses induced high levels of HA-specific immunoglobulin A (IgA) antibodies that were detected in bronchoalveolar lavages (BALs) and the serum. No mucosal responses could be detected after parenteral or intranasal immunization with naked-DNA. Furthermore, intranasal DNA vaccination with HA from a given H5N1 virus elicited full protection against the parental strain and partial cross-protection against a distinct highly pathogenic H5N1 strain that could be improved by adding neuraminidase (NA) DNA plasmids. Our observations warrant further investigation of intranasal DNA as an effective vaccination route.

Potency of a recombinant NDV-H5 vaccine against various HPAI H5N1 virus challenges in SPF chickens.

For the past decade, several recombinant Newcastle disease viruses (rNDV) have been used as a vector to express native or modified avian influenza (AI) hemagglutinins (HA) in order to give preventive protection against highly pathogenic avian influenza (HPAI) H5N1 viruses. Obtained protections were dependent on the age of the chickens, on the constructs and, in particular, on the homology between the HA that was inserted and the challenge strains. The objective of this study was to investigate the vaccine efficacy of a recombinant NDV La Sota-vectored vaccine expressing an Asian clade 1 H5 ectodomain (rNDV-H5) vaccine expressing a modified H5 ectodomain from an HPAI clade 1 H5N1 isolate as vaccine for 1-day-old specific-pathogen-free chickens. The inoculation route (oculonasal vs. drinking water), the dose-effect, and the protective range of this rNDV-H5 vaccine were studied. Both routes of vaccination induced an H5 serologic response and afforded a high degree of clinical protection against an Asian clade 1 HPAI H5N1 (AsH5N1) challenge without a significant difference between inoculation routes. A clear dose-effect could be demonstrated. Furthermore, when evaluating the protective range against antigenically divergent descendants of the Asian dade 1 HPAI H5N1 lineage, namely two Egyptian clade 2.2.1 H5N1 strains, the vaccine efficacy was less satisfactory. The rNDV-H5 vaccine provided good clinical protection and reduced viral shedding against Egyptian 2007 challenge but was unable to provide a similar protection against the more antigenically divergent Egyptian 2008 strain.

Immune responses and protection against H5N1 highly pathogenic avian influenza virus induced by the Newcastle disease virus H5 vaccine in ducks.

Ducks play an important role in the epidemiology of avian influenza, and there is a need for new avian influenza vaccines that are suitable for mass vaccination in ducks. The immune responses as well as highly pathogenic avian influenza (HPAI) H5N1 protection induced by a Newcastle disease virus (NDV) vector expressing an H5N1 hemagglutinin (rNDV-H5) were investigated in mule ducks, a hybrid between Muscovy (Cairina moschata domesticus) males and Pekin (Anas platyrhynchos domesticus) females. Immunological tools to measure NDV and H5-specific serum antibody, mucosal, and cell-mediated immune (CMI) responses in ducks have been validated after infection with the vector NDV and an H5N1 low pathogenic avian influenza virus. The effect of maternally-derived antibodies (MDAs) to NDV on the humoral and CMI responses after NDV-H5 vaccination was also investigated. Our results showed the rNDV-H5 vaccine elicits satisfactory humoral and cellular responses in 11-day-old ducks correlating with a complete clinical and virological protection against the H5N1 strain. However, vaccination with rNDV-H5 in the presence of NDV MDA induced lower NDV-specific serum antibody, mucosal, and CMI responses than in ducks with no MDA, while interestingly the H5-specific serum antibody and duodenal IgY response were higher in ducks with NDV MDA. To our knowledge, this is the first report of the use of an NDV vector in ducks and of an HPAI H5N1 challenge in mule ducks, which appeared to be as resistant as Pekin ducks.

Evaluation of four enzyme-linked immunosorbent assays for the serologic survey of avian influenza in wild bird species.

Wild birds that reside in aquatic environments are the major reservoir of avian influenza viruses (AIVs). Since this reservoir of AIVs forms a constant threat for poultry, many countries have engaged in AIV surveillance. More and more commercial enzyme-linked immunosorbent assays (ELISA) are available for serologic surveillance, but these tests are often developed and validated for use in domestic poultry. However, for a correct interpretation of ELISA test results from wild bird sera, more information is needed. In the present study, four ELISA test kits (ID-Vet IDScreen, IDEXX FlockChek AI MultiS-Screen Ab Test Kit, Synbiotics FluDETECTBE, and BioChek AIMSp) were compared for the serologic analysis of 172 serum samples from mallard, mute swan, and Canada goose. Samples were selected based on ID-Vet IDScreen results to obtain an approximately equal number of positive and negative samples. In addition, 92 serum samples from experimentally infected specific-pathogen-free (SPF) chickens and Pekin ducks were included in the tests for validation purposes. Cohen's kappa statistics and Spearman correlation coefficients were calculated for each combination of two tests and for each bird species. Test agreement for mallard sera varied from poor to moderate, while test results for Canada goose and swan sera agreed from fair to almost perfect. The best agreement was obtained with sera from experimentally infected SPF chickens and Pekin ducks. This study shows that some care must be taken before using nucleoprotein ELISAs for the testing of sera from wild birds and that more reliable validation studies should be considered before their use in the serologic surveillance of wild birds.

Different replication profiles in specific-pathogen-free chickens of two H7 low pathogenic avian influenza viruses isolated from wild birds.

During an active wild bird survey conducted in Belgium from 2007 to 2011, two low pathogenic avian influenza (LPAI) H7 viruses were isolated from wild birds: an H7N1 virus from a common shelduck (Tadorna tadorna) and an H7N7 virus from a Canada goose (Branta canadensis). The H7 sequence analyses and intravenous pathogenicity indices indicated that they were both low pathogenic isolates and genetically related to other recent European H7 LPAIs isolated from wild birds. Interestingly, the two isolates showed different replication profiles in specific-pathogen-free (SPF) chickens, but poultry can be at risk from both. Indeed, the H7N1 isolated from the common shelduck had the ability to infect and to replicate efficiently in SPF chickens as indicated by high oropharyngeal and cloacal excretions compatible with efficient transmission as well as strong immune responses. On the other hand, the H7N7 isolated from the Canada goose presented a lower replication profile because the inoculated chickens excreted less virus, mostly via the oropharyngeal route, and only three chickens seroconverted. None of the chickens showed clinical signs during the entire infection. Our study using an SPF chicken model underlines that the mechanisms of adaptation of LPAIs in poultry remain unpredictable and are still poorly understood but it represents a powerful tool to gain a better evaluation of the risks of LPAI circulation in poultry.

Risk assessment of SARS-CoV-2 infection in free-ranging wild animals in Belgium.

The aim of this review paper is to evaluate the putative susceptibilities of different free-ranging wild animal species in Belgium to SARS-CoV-2 and provide a risk assessment of SARS-CoV-2 infection in those animals. Since the onset of the COVID-19 pandemic, natural SARS-CoV-2 infections have mainly been confirmed in domestic and production animals, and in wild animals kept in captivity, although the numbers remain limited when compared to human cases. Recently, the first SARS-CoV-2 infections in presumably escaped minks found in the wild have been detected, further addressing the much-feared scenario of transmission of the virus to animals living in the wild and its consequences. Considering the most likely origin of the virus being a wild animal and the putative susceptibilities of free-ranging wild animal species to SARS-CoV-2, the risk of infection with possible establishment of the virus in these populations has to be investigated closely. The authors conclude that most attention should be given to surveillance and awareness-raising activities for SARS-CoV-2 infection in wild mustelids, bats, wild canids and felids, particularly these collected in wildlife rescue centres. People involved in frequent and close contact with wild animals should take all necessary precautionary measures to protect wild animals against exposure to the virus. More than one year after the first detection of SARS-CoV-2 in humans, the time has come to increase investments in research and surveillance activities in animals, including in free-ranging wild animals, as part of a One Health control of this pandemic. This study focussing on Belgium could be helpful for other countries with similar animal densities and ecosystems.